Traditional Bulgarian Fermented Foods as a Source of Beneficial Lactic Acid Bacteria.

BACKGROUND: Traditional Bulgarian fermented foods are prominent for their uniqueness of local ingredients, production methods, and endemic microbial species. The present research investigated the diversity and beneficial biological potential of lactic acid bacteria (LAB) isolated from various types of unique Bulgarian fermented foods. METHODS: Species identification was performed via 16S rDNA sequencing. Biological activity was evaluated by determining antibacterial activity (via agar well diffusion assay), H2O2 production, spectrophotometrically determined auto- and co-aggregation, microbial adhesion to hydrocarbon, and biofilm formation. The biosafety of the isolated lactic acid bacteria was established based on hemolytic activity and phenotypic and genotypic antibiotic susceptibility. RESULTS: Forty-five strains were isolated from fermented foods (sauerkraut, fermented green tomatoes, fermented cucumbers, kefir, white cheese, and Izvara (curdled milk)). Five species were detected: Lactiplantibacillus plantarum, Levilactobacillus koreensis, Levilactobacillus brevis, Lactobacillus helveticus, and Levilactobacillus yonginensis. The most prominent species was L. plantarum, at 47%. For the first time, L. koreensis and L. yonginensis, isolated from unique Bulgarian fermented foods, are reported in this study. The antibacterial effect of the cell-free supernatants was evaluated. An antagonistic effect was observed against Escherichia coli (57%) and Salmonella enterica subsp. enterica serotype Enteritidis (19%) for several L. plantarum strains. Only one L. brevis (Sauerkraut, S15) strain showed activity against E. coli. The best autoaggregation ability at hour 4 was observed for L. koreensis (fermented cucumbers, FC4) (48%) and L. brevis S2 (44%). The highest percentage of co-aggregation with Candida albicans, at hou 4 in the experiments, was observed for strains L. koreensis (fermented green tomato, FGT1) (70%), L. plantarum strains S2 (54%), S13 (51%), and S6 (50%), while at hour 24 for strains L. koreensis FGT1 (95%), L. brevis (Kefir, K7) (89%), L. plantarum S2 (72%), and L. koreensis FC2 (70%). Seven of the isolated LAB strains showed hydrophobicity above 40%. Our results showed that the ability of biofilm formation is strain-dependent. No hemolytic activity was detected. The antibiotic resistance to 10 antibiotics from different groups was tested phenotypically and genotypically. No amplification products were observed in any strains, confirming that the isolates did not carry antibiotic-resistance genes. CONCLUSIONS: Traditional fermented Bulgarian foods can be considered functional foods and beneficial LAB sources.

Involvement of oxidative stress in localization of bacterial spot infection in pepper plants.

Xanthomonas euvesicatoria is a major cause of bacterial spot disease in various crops. The present study was focused on the pathosystem pepper (Capsicum annuum L.) - X. euvesicatoria 269p (wild strain). The infectious process was studied using several different modes of in vivo inoculation under controlled conditions. The spread of the pathogen in different parts of the plants was monitored by a new qPCR procedure developed for the detection of X. euvesicatoria, as well as by re-isolation of viable bacterial cells. Photosynthesis, the number of viable pathogens, oxidative stress markers, activities of the main antioxidant enzymes, and levels of nonenzymatic antioxidants in the novel single-leaf model system were studied. The most important observation is that the invasion of the pathogen causes local infection and the dissemination of bacteria to the healthy parts of the host is blocked. The plants limit bacterial colonization around the entry points. Oxidative burst and alterations in antioxidant defenses are detected in infectious leaf lesions. Localized ROS overproduction resembles a hypersensitive response, but several differences can be observed. We assumed that pepper plants are more likely to manifest an intermediate phenotype, similar to lesions simulating disease or leaf flecking. By localizing the infection, possibly involving oxidative stress, the plant survives. However, the same applies to bacteria. The pathogen multiplies at the infection spots and is transmitted to other plants. Our conclusion is that the intermediate phenotype in the studied pathosystem is an example of long and successful co-evolution for both species.

Phytochemical Profile, Antioxidant Potential, Antimicrobial Activity, and Cytotoxicity of Dry Extract from Rosa damascena Mill.

Dry rose extract (DRE) obtained industrially by aqueous ethanol extraction from R. damascena flowers and its phenolic-enriched fraction, obtained by re-extraction with ethyl acetate (EAE) were the subject of this study. 1H NMR of DRE allowed the identification and quantitation of fructose and glucose, while the combined use of HPLC-DAD-ESIMS and HPLC-HRMS showed the presence of 14 kaempferol glycosides, 12 quercetin glycosides, 4 phenolic acids and their esters, 4 galloyl glycosides, 7 ellagitannins, and quinic acid. In addition, the structures of 13 of the flavonoid glycosides were further confirmed by NMR. EAE was found to be richer in TPC and TFC and showed better antioxidant activity (DPPH, ABTS, and FRAP) compared to DRE. Both extracts displayed significant activity against Propionibacterium acnes, Staphylococcus aureus, and S. epidermidis, but showed no activity against Candida albicans. Toxicity tests on normal human skin fibroblasts revealed low toxicity for both extracts with stronger effects observed at 24 hours of treatment that were compensated for over the following two days. Human hepatocarcinoma (HepG2) cells exhibited an opposite response after treatment with a concentration above 350 µg/mL for EAE and 500 µg/mL for DRE, showing increased toxicity after the third day of treatment. Lower concentrations were non-toxic and did not significantly affect the cell cycle parameters of either of the cell lines.

Cross-Over Pathogenic Bacteria Detected in Infected Tomatoes (Solanum lycopersicum L.) and Peppers (Capsicum annuum L.) in Bulgaria.

The ability of certain human pathogens to adapt to plants without losing their virulence toward people is a major concern today. Thus, the aim of the present work was the investigation of the presence of cross-over pathogenic bacteria in infected tomato and pepper plants. The objects of the study were 21 samples from seven different parts of the plants and three from tomato rhizosphere. In total, 26 strains were isolated, identified by MALDI-TOF, and phenotypically characterized. The PCR amplification of the rpoB gene was applied as an approach for the rapid detection of cross-over pathogens in plant samples. A great bacterial diversity was revealed from tomato samples as nine species were identified (Leclercia adecarboxylata, Pseudesherichia vulneris, Enterobacter cancerogenus, Enterobacter cloacae, Enterobacter bugandensis, Acinetobacter calcoaceticus, Pantoea agglomerans, Pantoea ananatis, and Pectobacterium carotovorum). Polymicrobial contaminations were observed in samples T2 (tomato flower) and T10 (tomato fruit). Five species were identified from pepper samples (P. agglomerans, L. adecarboxylata, Pseudomonas sp., Pseudomonas putida, and Enterococcus sp.). Antibiotic resistance patterns were assigned in accordance with EFSA recommendations. All isolates showed varying resistance to the tested antibiotics. The genetic basis for the phenotypic antibiotic resistance was not revealed. No genes for the virulence factors were found among the population. To our knowledge, this is the first overall investigation of tomato and pepper cross-over pathogenic bacterial populations in Bulgaria.

Broad host range bacteriophages found in rhizosphere soil of a healthy tomato plant in Bulgaria.

The urgent need of research of new approaches to control bacterial disease on economical important crops, focuses our attention on bacteriophages as alternative biocontrol agents. Thus, the purpose of this paper is to present the isolation and initial characterization of three bacteriophages (SfXv124t/1, 2 and 3) isolated from rhizosphere soil of a healthy tomato plant in Bulgaria that are capable to lyse three phytopathogenic bacteria. The initial characterization includes determination of: their host range, plaque morphology, optimal storage temperature of pure phage lysates, their sensitivity to UV light, thermal inactivation, optimal multiplicity of infection (MOI) and virion morphology. The obtained results showed that one of the phage isolates was capable to lyse wild strains from three phytopathogenic bacterial species: Xanthomonas vesicatoria, Xanthomonas euvesicatoria and Xanthomonas gardneri, and the two remaining phages were active against X. vesicatoria and X. euvesicatoria. On X. vesicatoria lawn, the phages produced the same plaque types that differed only in their size. Storage at 4 °C for 26 days did not lead to decrease in phage titer as opposed to storage at 28 °C followed by decrease to varying degree for all three phages. The results obtained after exposure of the phage lysates to sunlight (UVA + B) and UVC light in separate experiments showed that UVC had a potent phagocidal effect as after 50 min of exposure there were no viable phages in the samples. UVA an UVB had lethal effect for two of the phage isolates and absolutely no lethal effect for the third one as after 50 min of exposure to sunlight there was no decrease in the initial phage titer. Phage isolates were tested for their thermal inactivation after incubation of pure phage lysates at three different temperatures: 55 °C, 75 °C and 95 °C for a period of 10 and 30 min. The most lethal temperature turned out to be 95 °C as after 10 min there were no viable phages in the samples. Phage isolate SfXv124t/1 was the most susceptible as its titer decreased by 1 lg after 10 min of incubation at 55 °C and by another 1 lg after 30 min. The most thermally resistant isolate was SfXv124t/3 as its titer remained stable after 30 min of incubation at 55 °C and decreased only by lg after incubation at 75 °C for 10 min. The optimal MOI for SfXv124t/3 was 0,01 (tested range 0,01-100) with maximal phage titer, reported at the 24th hour of incubation. TEM micrographs of the same isolates reveals that it belongs to family Podoviridae.